AU2007280272A2 - Protein free formula - Google Patents

Protein free formula Download PDF

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Publication number
AU2007280272A2
AU2007280272A2 AU2007280272A AU2007280272A AU2007280272A2 AU 2007280272 A2 AU2007280272 A2 AU 2007280272A2 AU 2007280272 A AU2007280272 A AU 2007280272A AU 2007280272 A AU2007280272 A AU 2007280272A AU 2007280272 A2 AU2007280272 A2 AU 2007280272A2
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AU
Australia
Prior art keywords
composition
carbohydrate
protein
source
free
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Abandoned
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AU2007280272A
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AU2007280272A1 (en
Inventor
Catherine Teresa Deering
Sandra Helen Giffen
Jane Elizabeth Langford
Ian Sullivan
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SHS International Ltd
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Shs Int Ltd
SHS International Ltd
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Application filed by Shs Int Ltd, SHS International Ltd filed Critical Shs Int Ltd
Publication of AU2007280272A1 publication Critical patent/AU2007280272A1/en
Publication of AU2007280272A2 publication Critical patent/AU2007280272A2/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A23D9/013—Other fatty acid esters, e.g. phosphatides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
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    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/04—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by hydrolysis
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Description

WO 2008/015374 PCT/GB2007/002520 PROTEIN FREE FORMULA FIELD OF THE INVENTION The present invention relates to the stimulation of health in infants receiving an 5 amino acid based formula. BACKGROUND OF THE INVENTION In a preferred situation, infants are breastfed by the mother. However, often in infants, after an initial period of being breast fed, breastfeeding is stopped and the 10 infant diet comprises mainly infant milk formula. However in a small group of infants breastfeeding or the feeding of conventional infant formula results in adverse reactions, such as pain and allergic reaction. People suffering from an allergy may have difficulties in digesting or metabolizing some food 15 constituents, which then lead to gastro-intestinal and systemic allergic reactions. The allergic reactions are mainly directed to the protein fraction in the food. A significant change in the diet is often necessary to reduce adverse events. To prevent allergic reactions to protein, the infant food consists of hydrolysed milk 20 proteins, amino acids or non-milk proteins e.g. soy proteins and other nutritional components which do not cause allergic reactions. A known infant nutrition comprising amino acids as the nitrogen source is NeocateTM for use in patients suffering from allergy, gastro-intestinal complaints, eczema, 25 malabsorption or maldigestion. In the prior art the number of different sources of nutrients is often kept low to prevent introducing allergens in the formula by the addition of these ingredients. However, this has the disadvantage that infant also do not benefit from the beneficial effects of 30 such additional ingredients. W02005039319 discloses the use of synbiotics in infant formula including protein in hydrolysate form, thereby reducing the risk of allergy (page 11, line 35-37) 35 W02006091103 discloses a nutritional composition comprising synbiotics for the treatment and prevention of immune disorders including allergy. The use of protein hydrolysate and/or free amino acids for reducing the risk of allergy is disclosed. 1 WO 2008/015374 PCT/GB2007/002520 It is the aim of the present invention to improve the dietary formulas for this vulnerable patient group by providing additional nutritional benefits without introducing allergens in the composition. 5 SUMMARY OF THE INVENTION The present inventors have recognized that in an elemental formula containing a variety of free amino acids without intact protein, it is highly unpredictable how the intestinal flora develops. Instead of the bifidobacteria and lactobacilli dominant flora 10 in normal infants receiving breast milk, many other bacterial species, including potentially pathogenic species, may prevail in the intestine of infants receiving amino acid based foods. The development of a healthy intestinal flora is particularly important in all infants, as 15 these infants often already suffer from an impaired immunological function, resulting in colic, congestion, runny nose, wheezing, vomiting, diarrhoea, bloody stools, mucus in stools, rash, or eczema. The present inventors have recognized that the healthy flora development is of very 20 high importance in infants receiving a diet containing mainly free amino acid as a protein source. Without being bound by theory, the inventors recognized that a good flora, i.e. a flora rich in Bifidobacteria and Lactobacilli, is especially beneficial for the maturation of the (mucosal) immune system. A good flora prevents the development of allergies or at least reduces the severity of the allergies in such infants. Hence the 25 present inventors recognized the criticality of a beneficial flora development in these infants. In spite of the limitations in formulation of the dietary formula for such vulnerable infants, the present inventors found that a specific selection of dietary 30 oligosaccharides, preferably fructans and/or pectin degradation products can be beneficially added to the elemental formula to stimulate the flora development without causing any allergic side effects. No milk derived products can be used in this elemental formula, since this might lead 35 to the addition of milk allergens to the product. Therefore the prebiotic fibres according to this invention are carefully selected from the known prebiotic fibres in 2 WO 2008/015374 PCT/GB2007/002520 order to prevent the addition of milk or other intact and potential allergenic proteins that are present in the fibres to the non-allergic composition. The inventors surprisingly found in an internationally recognised model for allergy, 5 that when prebiotic fibres are combined with probiotic bacteria (preferably Bifidobacteria) a synergistic effect was present on the prevention of allergic reactions. This synergistic effect is potentially very beneficial to the infants, but could also be beneficial to adults. 10 BRIEF DESCRIPTION OF DRAWINGS Fig 1 shows the allergic response for various compositions tested in accordance with an Internationally recognised mouse vaccine model. 15 Fig 2 demonstrates the synergistic effect of TD1 (Bifidobacterium breve) and dietary fibers (OS) in a mouse casein allergy model. DETAILED DESCRIPTION OF THE INVENTION 20 A preferred embodiment according to the invention comprises the use of a composition comprising free amino acids as a sole source of protein, a fatty acid source comprising long chain polyunsaturated fatty acids, a carbohydrate source comprising digestible and indigestible carbohydrates, and milk protein free Bifidobacteria for the manufacture of a composition for treating a person suffering 25 from a. colic, congestion, runny nose, wheezing, vomiting, diarrhoea, bloody stools, mucus in stools, rash, eczema, gastroesophageal reflux, eosinophilic esophagitis or asthma ; b. cow's milk allergy and/or food protein intolerance; and/or 30 c. infections, wherein the indigestible carbohydrate is selected from a milk protein free source and the total composition is essentially free of intact proteins. The term "sole source of protein" as herein means that present composition 35 (preferably) contains at least 99 wt.% amino acids based on total protein, preferably at least 99.5, more preferably at least 99.9 wt.%. 3 WO 2008/015374 PCT/GB2007/002520 The invention further provides composition comprising a protein component, a fat component, a digestible carbohydrate component, a non-digestible carbohydrate and bifidobacteria wherein; a) the protein component contains more than 99 wt.% free amino acids 5 based on total protein, and comprising at least the following free amino acids: alanine, arginine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, valine and glutamine; b) the digestible carbohydrate component contain less that 2 wt.% lactose 10 based on total digestible carbohydrate; c) the non-digestible carbohydrate comprises soluble fructan with an average DP between 2 to 200 and a soluble galacturonic acid with an average DP between 2 and 200; and d) the fat component comprises between 0.1 and 5 wt% LCPUFA based 15 on total fatty acid content. As is shown in figure 1, the combination of long chain soluble fructans (LFOS) with an acid oligosaccharide (AOS), which was in this case pectin hydrolysate, work as well as the bifidobacteria when tested in the mouse model. It is clear however that if 20 the oligosaccharides are combined with the Bifidobacteria the effects are strongly improved. In a preferred embodiment the nutritional composition comprises both the non digestible fibres and the Bifidobacteria since this composition gives the best results in 25 the allergic mouse model. (see figure 1). Preferably Bifidobacteria breve is used. Non-digestible carbohydrate Fructans are fructose based neutral oligosaccharides (>50% of the monose units are fructose), preferably inulin, fructan and/or fructooligosaccharide, most preferably a 30 mixture of long chain fructooligosaccharide (IcFOS) with an average DP between 10 60 and short chain fructooligosaccharide (scFOS) with an average DP between 3 and 10. Preferred embodiment comprises a mixture of IcFOS and scFOS in a ratio of 1:9 since this ratio comes closer to the composition of oligosaccharides in breast milk and has been proven to effectively stimulate the growth of bifidobacteria in infants. 35 The present method preferably comprises the administration of a serving comprising between 0.05 and 25 grams non-digestible saccharide, preferably between 0.1 and 5 4 WO 2008/015374 PCT/GB2007/002520 grams. The present method preferably comprises the administration of a serving comprising between 0.05 and 25 grams scFOS, preferably between 0.1 and 5 gram scFOS. The present method preferably comprises the administration of 0.05 to 25 grams non-digestible saccharide per day, preferably between 0.1 and 5 grams per 5 day. Pectin degradation product Pectin is divided into two main categories: high methoxylated pectin, which is 10 characterized by a degree of methoxylation above 50% and low methoxylated pectin having a degree of methoxylation below 50%. As used herein, "degree of methoxylation" (also referred to as DE or "degree of esterification") is intended to mean the extent to which free carboxylic acid groups contained in the polygalacturonic acid chain have been esterified (e.g. by methylation). The present 15 pectin is preferably prepared from high methoxylated pectin. The pectin is preferably characterized by a degree of methoxylation above 20%, preferably above 30 % even more preferably above 50%. 20 The pectin as used in the present method has an average degree of polymerization (DP) between 2 and 500, preferably between 10 and 250 and most preferably between 20 and 50. When a mixture of pectin's with different degrees of polymerization is used, the average DP of the acid oligosaccharide mixture is preferably between 3 and 1000, more preferably between 3 and 250, even more 25 preferably between 3 and 50. It was found that a lower DP of the oligosaccharides improves the palatability and results in a reduced viscosity product if the acid oligosaccharide is administered in liquid form. The pectin is preferably administered in an amount of between 0.1 and 100 gram per 30 day, preferably between 0.4 and 50 grams per day, even more preferably between 1 and 20 gram per day. A preferred embodiment comprises fructans and pectin degradation products in a ratio 50:50 - 95: 5. Preferably the ratio is 85:15 since this is the ratio of neutral 35 oligosaccharides and acidic oligosaccharides as present in mother's milk. 5 WO 2008/015374 PCT/GB2007/002520 Probiotic bacteria The probiotic bacteria that are suitable for use in the invention should preferably have at least a positive effect in the mouse allergy model as described in the examples below. The probiotics further must be harvested in substantial absence of food 5 allergens. This requires special procedures or culture media without intact proteins for the preparation of the probiotic bacteria. Preferred embodiment therefore comprises probiotic bacteria in substantial absence of intact food proteins or food allergens, specifically in substantial absence of milk 10 proteins. In a preferred embodiment the probiotic bacteria are bifidobacteria. Even more preferred the probiotic bacterium is Bifidobacterium breve. Bifidobacteria have the strongest tolerance inducing effect in the mouse models used and are therefore 15 preferred. In particular Bifidobacterium breve is highly effective. In another preferred embodiment the Bifidobacteria are non-viable. This has the advantage that the shelf life of the product becomes longer and that the immune modulatory activity of the bacteria becomes independent of the number of live 20 bifidobacteria. Experiments have shown that immune stimulatory effects of non viable bacteria is similar and sometimes even better than the activity of live bifidobacteria. The present composition preferably comprises 102 to 1013 colony forming units (cfu) 25 of bifidobacteria per gram dry weight of the present composition, preferably 102 to 1012 cfu, more preferably 105 to 1010 cfu, most preferably from 10 4 to 5x1 09 cfu. Amino acids 30 Amino acids can be used in nutritional formula for babies and infants. However, the composition of the amino acids in the formula preferably comprises all essential amino acids except for patients with phenylketonuria (PKU) and non-PKU inborn errors of metabolism, and is preferably optimal for baby food or infant nutrition. The inventors than found that the most optimal amino acid composition should be as 35 close as possible to the amino acid composition of the protein fraction of mothers' milk. This results in a preferred amino acid composition as depicted in table 1 (see infra). 6 WO 2008/015374 PCT/GB2007/002520 In a preferred embodiment the composition comprises less than 1 wt.% peptides based on total protein and more than 99 wt.% free amino acids based on total protein, comprising at least the following free amino acids: alanine, arginine, aspartic 5 acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, valine and glutamine; Formula Formula according to the invention comprise free amino acids as nitrogen source, fat 10 including a fat blend comprising LCPUFA and carbohydrates. Vitamins and minerals are added according to legislation requirements. The present composition preferably provides nutrition to the infant, and comprises a lipid component, a protein component and a carbohydrate component. The lipid 15 component preferably provides 5 to 50% of the total calories, the protein component preferably provides 5 to 50% of the total calories, and the carbohydrate component preferably provides 15 to 90% of the total calories. The present composition is preferably used as an infant formula, wherein the lipid component provides 35 to 50% of the total calories, the protein component provides 7.5 to 12.5% of the total 20 calories, and the carbohydrate component provides 40 to 55% of the total calories. For calculation of the % of total calories for the protein component, the total of energy provided by the amino acids needs to be taken. LCPUFA 25 The content of LC-PUFA with 20 and 22 carbon atoms in the present composition, preferably does not exceed 15 wt.% of the total fat content, preferably does not exceed 10 wt.%, even more preferably does not exceed 5 wt.% of the total fat content. Preferably the present composition comprises at least 0.1 wt.%, preferably at least 0.25 wt, more preferably at least 0.5 wt.%, even more preferably at least 0.75 30 wt.% LC-PUFA with 20 and 22 carbon atoms of the total fat content. The Docosahexaenoic acid (DHA) content preferably does not exceed 5 wt.%, more preferably does not exceed 1 wt.%, but is at least 0.1 wt% of the total fat. As arachidonic acid (AA) was found to be particularly effective in reducing tight junction permeability, the present composition comprises relatively high amounts, preferably 35 at least 0.1 wt.%, even more preferably at least 0.25 wt.%, most preferably at least 0.5 wt.% of the total fat. The AA content preferably does not exceed 5 wt.%, more preferably does not exceed 1 wt.% of the total fat. Excess metabolites from AA may 7 WO 2008/015374 PCT/GB2007/002520 cause inflammation. Hence, the present composition preferably comprises AA and DHA, wherein the weight ratio AA/DHA preferably is above 0.25, preferably above 0.5, even more preferably above 1. The ratio is preferably below 25. 5 The LC-PUFAs are preferably non-fish derived single cell oils, e.g. available from Martek. Preferred features for compositions in accordance with the invention are set out in Tables 1-3. 10 Table 1. Amino acid content of composition according to the invention AA content as percentage of total AA Range (g/ 1ag) Amino Acid Units Infant 0 -1 year Infant 1-10 year Ala gm 3.95 4.01 3.12 3.03 Arg gm 6.99 7.02 14.24 13.91 Asp gm 6.54 6.57 5.73 5.59 Cys gm 2.59 2.58 1.85 2.91 Glu acid gm 0.00 0.00 0.00 Gly gm 6.15 6.12 5.11 5.01 His gm 4.02 3.99 3.71 3.61 Iso gm 6.15 6.12 5.11 5.01 Leu gm 10.56 10.51 8.54 8.32 Lys gm 7.19 7.19 6.25 6.11 Meth gm 1.68 1.69 4.02 3.90 Phe gm 7.51 7.47 7.10 6.93 Pro gm 4.73 4.72 5.97 5.82 Ser gm 4.60 4.61 3.71 3.61 thr gm 5.18 5.17 4.29 4.19 Try gm 2.07 2.08 1.72 1.69 tyr gm 4.73 4.72 1.44 1.40 Val gm 6.74 6.74 5.42 5.30 Car gm 0.06 0.06 0.10 0.12 tau gm 0.19 0.20 0.17 0.17 Glu gm 8.42 8.43 12.35 13.39 total gm 100 100 99.97 100 15 8 WO 2008/015374 PCT/GB2007/002520 Table 2. Nutritional infant formula for allergic infants Nutritional Profile of formula for infants according to the invention (powder) Units Level/100g Level/100kcal Protein Equivalent gm 13 2.9 Energy kcals 455 100 Nitrogen gm 2.1 Carbohydrate gm 49 10.8 Fat (total) gm 23 5.1 (MCT) % 33 (LCT) % 67 5 Table 3. Ranges in Long Chain Polyunsaturated (LCP) fatty acid composition % Fatty acids Preferred Range +/-25% Arachidonic acid 0.35 0.26 0.44 Docosahexanoic acid 0.20 0.15 0.25 Total LCP 0.55 0.41 0.69 The preferred ranges in the oligosaccharide composition are 0.4-1.2g/1 00ml wherein 85 w% is scFOS and IcFOS and 15 w% is pectin hydrolysate. Preferably the ratio 10 scFOS to lcFOS is between 2 and 20 even more preferably the ratio is 9. One preferred embodiment of milk protein and allergen free, nutritionally complete powdered formulation in accordance with the invention which is suitable for dilution with water to form an enteral feed comprises: 15 Component Per 1c00q Powder Protein equivalent (g) 13 Total amino acids (g) 15.5 Total fat (g) 23 20 Sunflower oil (g) 4 Fractionated coconut oil (g) 7 Canola oil (g) 4 High oleic sunflower oil (g) 6.6 ARASCOTM oil (g) 0.21 25 DHASCO TM oil (g) 0.11 9 WO 2008/015374 PCT/GB2007/002520 Carbohydrate: Maltodextrin (g) 49 Prebiotic (g) 5.33 ScFOS (g) 4.1 IcFOS (g) 0.43 5 A OS (g) 0.8 Probiotic: B Breve (colony forming units; CFU) I x 10 Other vitamins/minerals/trace elements Balance The above formulation provides approximately 455 kcal of energy per 1 OOg powder. 10 The formulation may be diluted with cooled, boiled water to provide a recommended concentration of 15% w/v. Uses 15 Preferable the composition is used for treating an infant (particularly with an atopic constitution) suffering from a. colic, congestion, runny nose, wheezing, vomiting, diarrhoea, bloody stools, mucus in stools, rash, eczema, gastroesophageal reflux, eosinophilic esophagitis or asthma; 20 b. cow's milk allergy and/or food protein intolerance; and/or c. infections The composition can also preferably be used to improve the stool characteristics of infants suffering from the above mentioned symptoms. The composition is 25 specifically designed for infants between 0-and 3 years. With some adaptations in the amino acid profile (see table 1) the composition is also suitable for infants between 3 and 10 years old. Allergic infants often suffer from diarrhoea but constipation also occurs. A preferred composition can be used to prevent and treat these symptoms comprises fibres according to the composition of mothers milk, wherein the ratio 30 scFOS/IcFOS is 9:1 and additionally pectin hydrolysate is present. EXAMPLES In order to determine the immune stimulating effects of oligosaccharides and probiotic Bifidobacterium breve (TD1) tests were performed in an internationally 35 recognized mouse vaccine model (fig 1) and in an allergy model (fig 2). 10 WO 2008/015374 PCT/GB2007/002520 Materials and method of the vaccine model Mice Female 6- and 8-week old C57BI/6JOlaHsd mice were obtained from Harlan (Horst, the Netherlands) and kept under normal conditions with a 12 h dark and light cycle 5 and free access to food and water. All experiments were approved by an independent animal experiments committee (DEC Consult, Bilthoven, The Netherlands). Diets and oligosaccharide preparations 10 All animals received semi-purified AIN-93G-based diets (Research Diet Services,Wijk bij Duurstede, The Netherlands). All supplemented oligosaccharide products were exchanged for the same amount of total carbohydrates, to keep this parameter equal. In addition, this approach resulted in a comparable overall carbohydrate composition in different diets, to ensure that the gut flora was minimally influenced by differences 15 between control and test diets in parameters such as gut passage time and fluid retention. The oligosaccharides were mixed into the AIN-93G diet and pressed into pellets. Vaccination protocol and DTH response 20 Vaccination experiments were performed using Influvac (Solvay Pharmaceuticals, Weesp, the Netherlands) from season 2002/2003. It is an inactivated influenza virus vaccine based on isolated haemagglutinin (HA) and neuraminidase antigens of three strains of myxovirus influenza, in a dose equivalent to 30 ug/mL HA per strain (90 pg/mL HA in total). An oil-adjuvant was used in all vaccinations (Stimune, previously 25 known as Specol; Cedi-diagnostics, Lelystad, The Netherlands). The mice received a primary vaccination and a booster vaccination, consisting of a subcutaneous (sc) injection of a 1:1 mix of vaccine and adjuvant in a total volume of 100 pL. The booster vaccination was given 21 days after the primary vaccination. The experiments ended 10 days after booster vaccination. Blood samples (taken by retro 30 orbital puncture) were taken before primary and secondary vaccination and at the end of the experiment. Negative control groups that were included in all experiments (indicated with 'sham group') received injections with a 1:1 mix of PBS and adjuvant in a total volume of 100 pL. Sham groups were never used for statistical comparisons to supplemented groups, but served solely to demonstrate the specificity of vaccine 35 induced responses. DTH reactions were induced 9 days after booster vaccination, by sc injection of 25 pL Influvac into the ear pinnae of both ears. Ear thickness was measured in duplicate before vaccine challenge and 24 h thereafter, using a digital 11 WO 2008/015374 PCT/GB2007/002520 micrometer (Mitutoyo Digimatic 293561, Veenendaal, The Netherlands). The DTH response was calculated by subtracting the basal ear thickness from the value at 24 h after challenge. 5 Material and methods of the cows milk protein allergy model Chemicals Casein and whey are obtained from DMV international, Veghel, The Netherlands. Cholera toxin is purchased from Quadratech Diagnostics, Epsom, UK. PBS is 10 purchased from Cambrex Bio Science, Verviers, Belgium. Elisa coating buffer is obtained from Sigma, Alphen aan den Rijn, The Netherlands. Biotin labeled rat anti mouse IgE is purchased from BD Biosciences, Alphen aan den Rijn, The Netherlands. All other chemicals are obtained from Sigma-Aldrich-Chemie, Zwijndrecht, The Netherlands. 15 Oral sensitization and challenge of mice Three- to 5-week-old specific pathogen free female C3H/HeOuJ mice (n = 4-6 per group) were purchased from Charles River Laboratories (Maastricht, the Netherlands), maintained on cow's milk protein free mouse chow (Special Diets 20 Services, Witham, Essex, UK) and housed in the animal facility at Utrecht University. Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Mice were intra gastric (i.g.) sensitized with 0.5 mL homogenized casein (40 mg/mL PBS) with cholera toxin (CT, 20 pL/mL PBS) as an adjuvant using a blunt needle. Control mice received CT alone or PBS. Mice were 25 boosted weekly for a period of 6 weeks, one week after the last sensitization mice were challenged i.g. with 100 mg casein. Blood samples were collected and centrifuged thereafter (15 min. at 13500 rpm.). Sera were stored at - 70 0 C. Mice were sacrificed by cervical dislocation half an hour after i.g. challenge. 30 Allergen specific skin response The acute allergen specific skin response was measured after injection of the specific protein in the ear pinnae. Before i.g. challenge (t=0), the control, casein sensitized mice were injected intra dermal (i.d.) in the left ear with 20 pL homogenized casein 35 (0.5 mg/mL in PBS) respectively. In the right ear 20 plL PBS was injected as a vehicle control. Also the CT and PBS mice received a casein ear challenge using PBS 12 WO 2008/015374 PCT/GB2007/002520 injections as control. Ear thickness was measured in duplicate using a digital micrometer (Mitutoyo, Veenendaal, The Netherlands), at 0, 1, 4 and 24 hour after challenge. The allergen specific ear swelling was calculated by subtracting the basal (0 h) and the control (right ear) thickness from the value measured at the three 5 different time points (1, 4 and 24 h). Measurement of serum immunoglobulin and mouse mast cell protease-1 levels Concentrations of total IgE and levels casein or whey specific IgE, IgG1 and igG2a were determined in serum of sacrificed mice by means of ELISA. Microlon plates 10 (Greiner, Alphen aan den Rijn, The Netherlands) were coated with casein in coating buffer or rat anti-mouse IgE (1 pg/mL) in PBS for 18 hours at 40C. Plates were washed and blocked for 1 hour with 5% BSA. Serum samples were applied in several dilutions and incubated for 2 hours at room temperature. Plates were washed 5 times and incubated with 1 pg biotin labeled rat anti mouse IgE for one and a half hour at 15 room temperature. After washing plates were incubated with horse radish peroxidase (HRP) for one hour, washed and developed with o-phenylendiamine (OPD). The reaction was stopped with 4M H 2
SO
4 and absorbance was measured at 490 nm on a Benchmark microplate reader (Biorad, California, USA). Serum concentrations of mouse mast cell protease-1 (mMCP-1) were determined as described previously 20 using a commercially available ELISA kit (Moredun Scientific Ltd., Midlothian, UK). The results of the mouse vaccine model are shown in Fig 1 from which it is clear that the combination of TD1 (= Bifidobacterium breve) provides the strongest effect on DTH response and is significantly better than TD1 alone or any other combination 25 tested. The results of the mouse casein allergy model are shown in Fig 2 and demonstrates that the combination of dietary fiber and TD1 synergistically inhibits the "allergenic" DTH response to casein. 30 13 In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to 5 preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the 10 common general knowledge in the art, in Australia or any other country. 1 3a 2171229_1 (GHMatters) 28/1/10

Claims (18)

1. Use of a composition comprising free amino acids as a sole source of protein, a fatty acid source comprising long chain polyunsaturated fatty acids, a carbohydrate 5 source comprising digestible and indigestible carbohydrates, and milk protein free Bifidobacteria for the manufacture of a composition for treating a person suffering from a. colic, congestion, runny nose, wheezing, vomiting, diarrhoea, bloody stools, mucus in stools, rash, eczema, gastroesophageal reflux, eosinophilic esophagitis or asthma; 10 b. cow's milk allergy and/or food protein intolerance; and/or c. infections, wherein the indigestible carbohydrate is selected from a milk protein free source and the total composition is essentially free of intact proteins. 15
2. Use according to claim 1 wherein the person is an infant in the age between 0 and 36 months.
3. Use of a composition comprising free amino acids as a sole source of amino acids, a fatty acid source comprising long chain polyunsaturated fatty acids, a 20 carbohydrate source comprising digestible and indigestible carbohydrates, and milk protein free Bifidobacteria for the manufacture of a composition for stimulating the maturation of the immune system in an infant with an atopic constitution, wherein the indigestible carbohydrate is selected from a milk protein free source and the total composition is essentially free of intact proteins. 25
4. Use according to any of claims 1 to 3 wherein free amino acids based on total protein, comprising at least the following free amino acids: alanine, arginine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, valine and glutamine. 30
5. Use of a composition according to any of the claims 1-4 wherein the amino acids comprise between 5 - 16en% of the total composition, between 30 - 60 en% fat, whereof between 0.1 - 5en% LCPUFA and between 25 - 75 en% digestible carbohydrates. 35 14 21712291 (GHMatters) 28/1/10
6. Use according to claim 5 wherein the LCPUFA comprise between 0.2-1 en% of the total composition.
7. Use according to any of claims 1-6 wherein the indigestible carbohydrate 5 comprises (i) soluble fructan with an average degree of polymerisation between 3 to 200 and (ii) a soluble galacturonic acid with an average degree of polymerisation between 3 and 200.
8. Composition comprising a protein component, a fat component, a digestible 10 carbohydrate component, a non-digestible carbohydrate and milk protein free Bifidobacteria wherein; (a) the protein component comprises free amino acids as the sole source of protein and comprises at least the following free amino acids: alanine, arginine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, 15 lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, valine and glutamine; (b) the digestible carbohydrate component contain less than 2 wt.% lactose based on total digestible carbohydrate; (c) the non-digestible carbohydrate comprises soluble fructan with an 20 average DP between 2 to 200 and a soluble galacturonic acid with an average DP between 2 and 200; and (d) the fat component comprises between 0.1 and 5 wt.% LCPUFA based on total fatty acid content. 25
9. Composition according to claim 8 wherein: the fat component comprises between 0.2 and 1 wt.% LCPUFA based on the total fatty acid content. 30
10. Composition according to claim 8 or 9, comprising non-digestible, soluble fructan with an average DP between 2 to 200 and a non-digestible, soluble galacturonic acid with an average DP between 2 and 200.
11. Composition according to any one of claims 8 to 10, comprising Bifidobacterium 35 breve. 15 2171229_1 (GHMatters) 28/1/10
12. Composition according to any of claims 8-11, comprising arachidonic acid and/or docosahexaenoic acid.
13. Composition according to any of claims 8-12, wherein the indigestible 5 carbohydrate consist of long chain FOS and short chain FOS.
14. Composition according to claim 13 wherein the long chain FOS has an average DP between 10-60 and the short chain FOS has an average DP between 3 and 10. 10
15. Composition according to any of claims 8-14 wherein the product is a powder or a liquid.
16. A composition comprising free amino acids as a sole source of protein, a fatty acid source comprising long chain polyunsaturated fatty acids, a carbohydrate source 15 comprising digestible and indigestible carbohydrates, and milk protein free Bifidobacteria wherein the indigestible carbohydrate is selected from a milk protein free source and the total composition is essentially free of intact proteins for use in treating a person suffering from (a) colic, congestion, runny nose, wheezing, vomiting, diarrhoea, bloody 2 0 stools, mucus in stools, rash, eczema, gastroesophageal reflux, eosinophilic esophagitis or asthma; (b) cow's milk allergy and/or food protein intolerance; (c) and/or infections. 25
17. A method of treating a person suffering from (a) colic, congestion, runny nose, wheezing, vomiting, diarrhoea, bloody stools, mucus in stools, rash, eczema, gastroesophageal reflux, eosinophilic esophagitis or asthma; (b) cow's milk allergy and/or food protein intolerance; 3 0 (c) and/or infections. comprising administering to that person a composition comprising free amino acids as a sole source of protein, a fatty acid source comprising long chain polyunsaturated fatty acids, a carbohydrate source comprising digestible and indigestible 35 carbohydrates, and milk protein free Bifidobacteria wherein the indigestible 16 rrorl Unknown doc nt property neme. 28/1/10 carbohydrate is selected from a milk protein free source and the total composition is essentially free of intact proteins.
18. A use according to claim 1 or claim 3 a composition according to claim 8 or 5 claim 16 or a method according to claim 17, substantially as hereinbefore described, with reference to the examples, and, or figures. 1 6a ErrorI Unknown document property name. 28/1/10
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